PROJECT SUMMARY Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta-cells. Nonobese diabetic (NOD) mice develop spontaneous T1D and have been used extensively to study the genetic and pathogenic mechanisms of this autoimmune disease. Recently, a diabetogenic role of interleukin (IL)-21 has emerged, and higher levels of this cytokine were shown to be expressed by NOD mice compared to the T1D resistant strains. IL-21 is produced by CD4 T cells and its contribution to T1D is in part through its ability to support the survival and function of diabetogenic CD8 T cells that directly kill pancreatic beta-cells. NOD mice have an age dependent accumulation of IL-21+ CD4 T cells in islets. Importantly, NOD mice genetically rendered IL-21-deficient are completely resistant to the development of insulitis and diabetes. Interestingly, NOD islet IL-21+ CD4 T cells have a unique phenotype distinct from that of typical Th17 and Tfh CD4 T cell effector subsets known as the main IL-21 producers. Consistent with a diabetogenic function of IL- 21 in NOD mice, available evidence also supports its role in human T1D. Significantly higher levels of circulating IL-21+ CD4 T cells and IL-21 protein were found in subjects with beta-cell autoimmunity than in healthy controls. How IL-21+ CD4 T cells emerge and accumulate during the progression of T1D in both NOD mice and humans remains largely unknown and therefore deserves further investigation. More than 50 loci have been associated with human T1D by genome wide association studies (GWAS). One of the top risk genes is PTPN22. The human PTPN22 risk allele modulates T cell functions and promotes T1D when introduced into NOD mice. Notably, PTPN22 deficiency on the non-autoimmune prone C57BL/6 mouse strain background has been associated with a higher frequency of Tfh CD4 T cells and their enhanced production of IL-21. Addressing whether PTPN22 regulates diabetes development through modulating the differentiation states of islet IL-21+ CD4 T cells in NOD mice will provide a better understanding of the genetic control of T1D. Our hypothesis is that the ability of beta-cell autoreactive CD4 T cells to produce IL-21 represents a unique differentiation state that is modulated by Ptpn22 variants. We propose the following aims to test this hypothesis. Aim 1. To define the differentiation state of islet-infiltrating IL-21-expressing CD4 T cells by identifying their transcriptional networks using single-cell RNA sequencing. Aim 2. To determine the effects of Ptpn22 variants on the differentiation of islet-infiltrating IL-21-expressing CD4 T cells.